Mitragyna speciosa (kratom) has exploded onto the US market, with an estimated 55,000 kg entering the US between 2014 and 2016, corresponding to 12 million kratom doses and one million users. The FDA recently used computational modeling to predict that 22 kratom constituents bind to opioid receptors; however, these predictions await translation into whole animals. There is an urgent need for systematic, pharmacological testing of kratom alkaloids to help inform abuse risk. We feel it is important to assess the behavioral effects of not only individual alkaloids, but also alkaloid mixtures representative of plant material in commercially used products. We will extract, isolate, and purify alkaloids from two kratom sources: dried leaf material of Mitragyna speciosa trees and a US commercial product (i.e., OPMS Gold capsules). We will quantify up to ten kratom alkaloids simultaneously using our bioanalytical methods. In a pharmacokinetic-based Aim 1, we will quantify kratom alkaloids in multiple biological matrices (urine, plasma, brain, liver) after p.o. and i.v. administration in rats to obtain a comprehensive ADME profile. Liver microsomes, recombinant CYP450 enzymes, and specific chemical CYP450 inhibitors will be used to identify pathways of metabolism and biologically active metabolites. In our pharmacodynamic-based Aim 2, we will use drug discrimination to identify receptor mechanism(s) underlying abuse-related effects. Four separate groups of rats will be trained to discriminate one of the following: 1) alkaloid mixture in proportion to kratom dried leaf material, 2) alkaloid mixture in proportion to commercially available kratom product (both containing 32 mg/kg mitragynine, 3) 32 mg/kg mitragynine alone, or 4) 3.2 mg/kg 7- hydroxymitragynine alone. Moreover, we will use i.v. drug self-administration to assess abuse risk, and to assess how kratom alkaloids modify the reinforcing effects of abused opioids (i.e., heroin). Male and female rats will be used throughout to assess sex as a biological variable. The overarching hypothesis is that Mitragyna speciosa has a complex pharmacology resulting from multiple alkaloids differentially interacting with both opioid and non- opioid receptors. The following specific hypotheses will be tested: 1) alkaloids interact (i.e., exert synergistic and antagonist effects) with each other and with other abused opioids in drug discrimination and self-administration assays; 2) some of the interactions are due to PK; 3) behavioral effects of the parent alkaloid are due in part to both the parent compound and its behaviorally active metabolites; 4) no single alkaloid accounts for the discriminative stimulus and reinforcing effects of the mixtures; and 5) both opioid and adrenergic receptors mediate the effects of alkaloid mixtures representative of natural plant material. Subtraction and addition of individual alkaloids will help identify the alkaloids most responsible for kratom's integrated pharmacology. After 5 years, we expect to demonstrate that kratom alkaloids exert differential effects through multiple receptor systems, and we further expect to identify key pharmacological mechanisms responsible for the widespread use of kratom.